1. Field of the Invention
The invention disclosed herein relates to methods and compositions for the efficient production of bacterial plasmid-based shuttle vectors free of linear plasmid-derived DNA contaminants, and particularly such vectors as might be used in DNA vaccination and gene therapy.
2. Description of the Related Art
The application of recombinant DNA technology to eukaryotic cells has come to rely on shuttle vectors, plasmids that can be readily produced in bacterial culture but include elements that function in eukaryotic cells. Generally such plasmids contain a bacterial origin of replication and a selectable marker. In most cases the origin of replication is a high copy number origin so that many copies of the plasmid can replicate simultaneously in each bacterial cell thereby providing higher yields in production. The most commonly used origins of replications include pMB1, ColE1, and pUC. These origins are closely related and can be found in high and low copy number variants. The selectable marker serves to ensure that only bacteria containing the plasmid survive in culture. The eukaryotic elements most often consist of a viral or eukaryotic promoter linked to a protein coding frame leading to production of a peptide or protein when the plasmid is transduced into an appropriate eukaryotic host cell. In other cases transcription from the promoter accomplishes expression of a viral genomic RNA, leading to production of virus particles, or an antisense RNA. Various other elements may be included depending on the functions sought and these functions may dictate the arrangement of the elements involved. However no rules have been described for the arrangement of functionally unrelated elements.